Enrico Fainardi

Following the Fifth International Conference on non-classical HLA-G antigens (HLA-G), held in Paris in July 2009, we selected some topics which focus on emerging aspects in the setting of HLA-G functions. In particular, HLA-G molecules could play a role in: (1) various inflammatory disorders, such as multiple sclerosis, intracerebral hemorrhage, gastrointestinal, skin and rheumatic diseases, and asthma, where they may act as immunoregulatory factors; (2) the mechanisms to escape immune surveillance utilized by several viruses, such as human cytomegalovirus, herpes simplex virus type 1, rabies virus, hepatitis C virus, influenza virus type A and human immunodeficiency virus 1 (HIV-1); and (3) cytokine/chemokine network and stem cell transplantation, since they seem to modulate cell migration by the downregulation of chemokine receptor expression and mesenchymal stem cell activity blocking of effector cell functions and the generation of regulatory T cells. However, the immunomodulatory circuits mediated by HLA-G proteins still remain to be clarified.

Thrombolysis depends on the ability of blood and thrombolytic agents to permeate thrombus. We devise a novel technique to quantify blood permeating through thrombi and determine whether this parameter predicts early recanalization with intravenous tissue-type plasminogen activator. Intravenous tissue-type plasminogen activator-treated patients with stroke and complete occlusion on computed tomographic angiography were analyzed using perfusion computed tomography and a delay insensitive algorithm. We generated maps that measure delay in arrival time of contrast within the intracranial arterial tree (T0 maps). A positive sloped regression line of T0 values measured along artery silhouette distal to thrombus was defined as marker of permeable thrombus (occult anterograde flow). Median T0 values at proximal and distal thrombus interface were measured. Early recanalization was assessed on first angiography of subsequent intra-arterial procedure or on a 4-hour computed tomographic angiogr...

In this study, neoplastic perfusion abnormalities were investigated by computed tomography perfusion (CTP) scanning in 38 patients with solitary intra-axial brain tumors (19 with high grade gliomas, 7 with low grade gliomas and 12 with brain metastasis). Regional cerebral blood flow (rCBF), cerebral blood volume (rCBV), mean transit time (rMTT) and permeability surface flow (rPSF) levels were measured in two different regions of interest: (1) enhancing or non-enhancing tumor tissue and (2) a mirror area of apparently normal brain tissue located in the contralateral hemisphere. rCBF mean levels were greater in tumoral tissue than in the contralateral area for high-grade gliomas (p < 0.02). rCBV and rPSF mean values were higher in tumoral tissue than in the contralateral area for high-grade gliomas (p < 0.01 and p < 0.05, respectively) and metastasis (p < 0.05 and p < 0.001, respectively). rCBV mean values of tumoral tissue were greater in high-grade than in low-grade gliomas (p < 0.05). rPSF mean levels of tumoral tissue were higher in metastasis than in low-grade gliomas (p < 0.02). These findings indicate that multi-parametric CTP mapping may contribute to differential diagnosis of solitary intra-axial brain tumors.

We designed a large multicentric study to analyse the presence of MSRV particles in blood and CSF of a large cohort of patients and controls from different European areas. 149 MS patients and 153 neurological and healthy controls were selected from Sardinia, Spain, Northern-Italy and Sweden. To avoid biological and inter-assay variability MSRV was detected within a single laboratory through nested and real-time PCR assays specific for pol and env genes. MSRV detection in blood and CSF of MS patients and controls in populations of different ethnicity gave significant differences (p<0.05 compared to neurological controls and <0.001 compared to healthy controls). The presence and viral load of MSRV are significantly associated with MS as compared to neurological and healthy controls in all ethnic groups.

Cerebrospinal fluid (CSF) levels of sHLA-G (sHLA-G1/HLA-G5) molecules and their soluble isoforms HLA-G5 and sHLA-G1 were measured by ELISA procedures in 68 relapsing–remitting Multiple Sclerosis (RR MS) patients, in 67 patients with other inflammatory neurological disorders (OIND) and in 70 subjects with non-inflammatory neurological disorders (NIND). CSF concentrations of sHLA-G1/HLA-G5 and HLA-G5 were higher in RR MS than in OIND

It has recently become clear that interferon-β (IFN-β) treatment is effective in ameliorating relapsing–remitting multiple sclerosis (RRMS) through an as yet unidentified mechanism. As there is no recognisable biological indicator to predict responsiveness to IFN-β treatment, we have investigated fluctuations in serum sHLA-I levels in MS patients undergoing IFN-β 1b therapy. Serum sHLA-I concentrations measured by enzyme-linked immunosorbent assay (ELISA)

Serum levels of sHLA-G (sHLA-G1/HLA-G5) antigens and their soluble isoforms, sHLA-G1 and HLA-G5, were measured by ELISA in 22 patients with spontaneous intracerebral hemorrhage (SICH) at 24 h, 48 h and 7 days after bleeding. The perihematomal edema volume was calculated on non-enhanced computed tomography scans using the formula AxBxC/2 at the same time points. The mean serum concentrations of sHLA-G1/HLA-G5 and sHLA-G1 as well as the perihematomal edema volume changed significantly over time (p < 0.0001, p < 0.001 and p < 0.0001, respectively), whereas no statistical differences were found in serum HLA-G5 concentrations over the course of the experiment. In comparison to the values found at 24 h, sHLA-G1/HLA-G5 and sHLA-G1 increased at 48 h and then decreased at 7 days, whereas the perihematomal edema volume was more elevated at 48 h and, to a lesser extent, at 7 days. A positive correlation was detected between mean serum sHLA-G1/HLA-G5 and sHLA-G1 levels and perihematomal edema volume at 24 h (p < 0.02) and at 48 h (p < 0.01). Our results may indicate a role for sHLA-G in inflammatory mechanisms related to SICH, where these proteins probably act as anti-inflammatory molecules and are predominantly produced as the sHLA-G1 isoform.

Traumatic subarachnoid hemorrhage (tSAH) is a frequent finding after closed-head injuries, and its presence is a powerful factor associated with poor outcome. The exact mechanism linking tSAH and an adverse outcome is poorly understood. The aim of this study was to identify the factors that may predict outcomes and changes in the computed tomographic (CT) scans of lesions in a selected population of tSAH patients. We evaluated 141 patients admitted consecutively from January 1, 1997, to January 31, 1999, with a CT diagnosis of tSAH. The admission and "worst" CT scans were recorded. CT scan changes were reported as "significant CT progression" (changes in the Marshall classification) or "any CT progression." The amount of subarachnoid blood was recorded using a modified Fisher classification. Outcome was assessed at 6 months after injury with the Glasgow Outcome Scale. Twenty-eight patients (19.9%) had an unfavorable Glasgow Outcome Scale outcome. In the univariate analysis, prognosis was significantly related to age, admission Glasgow Coma Scale score, Marshall CT classification score at admission and on the worst CT scan, amount of tSAH, and volume of the associated brain contusions. From multivariate analysis, the only factors independently related to outcome were the Glasgow Coma Scale score (P < 0.01) and size of the tSAH at admission (P < 0.001). Thirty-four patients (24.1%) had significant CT lesion progression, and 66 patients (46.8%) had some lesion progression. Patients having significant progression of the lesion had a higher risk of an unfavorable outcome (32 versus 10%; P = 0.004). Unadjusted factors predicting CT progression were the Glasgow Coma Scale score at admission, the Marshall classification at admission, the amount of subarachnoid blood, and the presence or volume of associated brain contusions at admission. Independent factors associated with significant CT progression were the amount of tSAH (P < 0.001) and the presence or volume of brain contusions at admission (P < 0.001). The outcome of patients with tSAH at admission is related in a logistic regression analysis to the admission Glasgow Coma Scale score and to the amount of subarachnoid blood. These patients also have a significant risk of CT progression. The amount of subarachnoid blood and the presence of associated parenchymal damage are powerful independent factors associated with CT progression, thus linking poor outcomes and CT changes.

To evaluate the response to an acute elevation of cerebral perfusion pressure (CPP) of the regional cerebral blood flow (rCBF) measured in the edematous area of traumatic contusions. rCBF was measured in the intracontusional low-density area, in the pericontusional healthy-appearing brain tissue surrounding the contusion, in a healthy-appearing area in the contralateral hemisphere, in 16 head-injured patients with 16 traumatic contusions larger than 2 cm at baseline, and after 20 minutes of norepinephrine-induced 20-mmHg elevation of CPP levels. After an induced acute elevation of CPP from baseline values of 65.8 ml/100 g/min (standard deviation, 8.6) to final values of 88.7 ml/100 g/min (standard deviation, 8.9; P < or = 0.0001), we found that rCBF mean levels decreased in the intracontusional low-density area (P = 0.0278), and change in rCBF was inversely associated to the baseline values. After grouping contusions according to the rCBF response to induced acute CPP elevation, rCBF mean values recorded at baseline were significantly lower in lesions with "rCBF improvement" than in those with "rCBF reduction" in the intracontusional low-density area (P = 0.0435). Our findings suggest that CPP elevation induced by norepinephrine is effective in improving contusional rCBF only in selected cases, which are represented by a subset of contusions with critical perfusion, which can be identified by rCBF measurements. Conversely, in contusions with rCBF higher than critical low values, the CPP elevation could probably induce a temporary breakdown of the blood brain barrier, and the norepinephrine leads to a vasoconstriction with a worsening of regional perfusion.

To verify the values and the time course of regional cerebral blood flow (rCBF) in the cortex located beneath an evacuated acute subdural hematoma (SDH) and their relationship with neurological outcome. rCBF levels were measured in multiple regions of interest, by means of a Xe-computed tomographic technique, in the cortex underlying an evacuated SDH and contralaterally in 20 patients with moderate or severe traumatic brain injury and an evacuated acute SDH. Twenty-three patients with moderate or severe traumatic brain injury and an evacuated extradural hematoma or diffuse injury served as the control group. Outcome was evaluated by means of the Glasgow Outcome Scale at 12 months. Values for the maximum (rCBFmax) and the mean of all rCBF levels in the cortex beneath the evacuated SDH were more frequently consistent with hyperemia. The side-to-side differences in the mean of all rCBF and rCBFmax levels between lesioned and nonlesioned hemispheres were greater in patients with evacuated SDH than in controls (P = 0.0013 and P = 0.0018, respectively). The side-to-side difference in the maximum rCBF value was higher in SDH patients with unfavorable outcomes than in controls at 24 to 96 hours and at 4 to 7 days and higher than in patients with favorable outcomes at 4 to 7 days. The widest side-to-side difference in rCBFmax value was more elevated in patients with an evacuated SDH with unfavorable outcome than in patients with a favorable outcome (P = 0.047), whereas no differences were found in controls. The SDH thickness and the associated midline shift were greater in patients with unfavorable outcomes than in those with favorable outcomes. On average, hyperemic long-lasting rCBF values frequently occur in the cortex located beneath an evacuated SDH and seem to be associated with unfavorable outcome.

Low-grade gliomas are slow-growing tumors invading eloquent areas and white matter pathways. For many decades these tumors were considered inoperable because of their high tropism for eloquent areas. However, the young age of the patients and the inescapable anaplastic transformation have recently suggested more aggressive treatments. We analyzed the neurological and neuro-oncological outcome of 12 patients who underwent surgery fully awake for the resection of LGG, harboring eloquent areas. 10 right- and 2 left-handed patients underwent pre-operative assessment: Karnofsky Performance Status, Edinburgh Handedness Inventory Score; neuropsychological and neurophysiological evaluations, according to the tumor location. During surgery we performed: sensory-motor-evoked potentials, continuous electro-corticography and bipolar/monopolar cortico-subcortical mapping during neuropsychological tests. The resection rate was calculated with neuro-imaging elaboration software. No permanent post-operative deficits were reported; 2 patients improved after surgery. No impairment of cognitive functions was reported. The KPS improved in 8 patients and was steady in the others. The mean resection rate was 78.3%. The resection allowed the control of pre-operative seizures without increasing the drug intake. Awake surgery allowed a good resection rate despite the eloquent location of the tumors, without post-operative deficit. The neuropsychological outcome was unchanged after surgery. The resection seems to improve seizure control. All the patients came back to normal life and work. In conclusion, awake surgery is reliable and feasible in removal of LGG, even if invading the main eloquent areas and networks. All the patients experienced a normal life after surgery, without permanent deficits.

The aim of this study was to provide further insight into the effective contribution of classical soluble HLA-A, B and C class Ia (sHLA-I) and non-classical soluble HLA-G class Ib (sHLA-G) molecules in immune dysregulation occurring in multiple sclerosis (MS). We evaluated by enzyme-linked immunosorbent assay (ELISA) technique intrathecal synthesis and cerebrospinal fluid (CSF) and serum levels of sHLA-I and sHLA-G in 69 relapsing-remitting (RR), 21 secondary progressive (SP) and 13 primary progressive (PP) MS patients stratified according to clinical and magnetic resonance imaging (MRI) evidence of disease activity. We also tested, as neurological controls, 91 patients with other inflammatory neurological disorders (OIND) and 92 with non-inflammatory neurological disorders (NIND). Eighty-two healthy volunteers served as further controls for sHLA-I and sHLA-G determinations. An intrathecal production of sHLA-I and sHLA-G detected by specific indexes was significantly more frequent in MS patients than in controls (P<0.01). An intrathecal synthesis of sHLA-I was prevalent in clinically (P<0.02) and MRI active (P<0.001) MS, whereas a CSF-restricted release of sHLA-G predominated in clinically (P<0.01) and MRI stable (P<0.001) MS. sHLA-I levels were low in the serum of clinically active (P<0.001) and high in the CSF of MRI active (P<0.01) MS. Conversely, sHLA-G concentrations were decreased in the serum of clinically stable MS (P<0.01) and increased in the CSF of MRI inactive MS (P<0.001). The trends towards a negative correlation observed between CSF and serum concentrations and intrathecal synthesis of sHLA-I and sHLA-G in patients without evidence of clinical and MRI activity confirmed that intrathecal production and fluctuations in CSF and serum concentrations of sHLA-I and sHLA-G were reciprocal in MS. Our results suggest that, in MS, a balance between classical sHLA-I and non-classical sHLA-G products modulating both MRI and clinical disease activity in opposite directions may exist.

In this study, we employed a sensitive activity assay system to measure cerebrospinal fluid (CSF) and serum levels of active matrix metalloproteinase-9 (MMP-9) in 37 relapsing-remitting (RR), 15 secondary progressive (SP) and nine primary progressive (PP) multiple sclerosis (MS) patients, grouped according to clinical and magnetic resonance imaging (MRI) evidence of disease activity. We also studied, as neurological controls, 48 patients with other inflammatory neurological disorders (OIND) and 48 with non-inflammatory neurological disorders (NIND). To assess active MMP-9/TIMP-1 circuit, CSF and serum levels of MMP-9 tissue inhibitor TIMP-1 were quantified by ELISA in the same patient population. CSF mean levels of active MMP-9, CSF active MMP-9/TIMP-1 ratios and intrathecal active MMP-9 synthesis, as indicated by specific index, were more elevated in MS than in NIND (P < 0.05, < 0.02 and < 0.02, respectively), serum active MMP-9/TIMP-1 ratio was higher in MS (P < 0.01) and OIND (P < 0.02) than in NIND, and serum TIMP-1 concentrations were lower in MS than in NIND (P<0.05). More importantly, serum active MMP-9 mean levels, serum active MMP-9/TIMP-1 ratio and intrathecal production of active MMP-9 were increased in MS patients with clinical (P < 0.001, < 0.001 and < 0.05, respectively) and MRI (P < 0.001, < 0.001 and < 0.02, respectively) disease activity, whereas CSF mean concentrations of active MMP-9 and CSF active MMP-9/TIMP-1 ratio were enhanced only in MS patients with MRI evidence of disease activity (P < 0.02 and < 0.01, respectively). Altogether, these findings suggest that a shift in MMP-9/TIMP-1 balance towards proteolytic activity of MMP-9 could be relevant in MS immune dysregulation. In addition, our results indicate that CSF and serum levels of active MMP-9 may represent a potential surrogate biomarker for monitoring MS disease activity. In particular, serum active MMP-9/TIMP-1 ratio seems to be a very appropriate indicator of ongoing MS inflammation, since it is easily measurable.

JC virus (JCV), the causative agent of progressive multifocal leukoencephalopathy (PML), has been proposed as a possible aetiopathogenic factor in multiple sclerosis (MS). We performed a study to search the LT region of JCV genome by nested PCR in cerebrospinal fluid (CSF), peripheral blood mononuclear cell (PBMC) and urine samples collected from 121 MS patients, 24 patients with other neurological disorders (OND), 30 non neurological patients (NND) and in PBMCs and urine of 40 healthy subjects. JCV DNA has been found in the CSF of 11 MS patients (9%) while all the CSFs from the 24 OND and the 30 NND cases were negative. No significant differences have been observed as regard to the frequency of JCV DNA detection in PBMCs and urine between the MS patients and the control groups. Nucleotide sequences analysis of seven JCV CSF isolates showed that five strains were identical the prototypal strain, while the other two had a base mutation (T-->C) in 4286 nucleotide (nt). The finding of JCV DNA in the CSF of MS patients suggest that JCV could play a role in the triggering and/or in the maintenance of MS aetiopathogenic process, and therefore it should be taken in consideration when monitoring this disease.

Cerebrospinal fluid (CSF) concentrations of soluble human leukocyte antigen class I (HLA-I) (sHLA-I), HLA-G (sHLA-G) and anti-apoptotic Fas (sFas) molecules were measured by enzyme linked immunosorbent assay technique in 65 relapsing-remitting (RR) MS patients classified according to clinical and magnetic resonance imaging (MRI) evidence of disease activity. Sixty-four patients with other inflammatory neurological disorders (OIND) and 64 subjects with noninflammatory neurological disorders (NIND) served as controls. CSF concentrations were higher in RRMS and in OIND than in NIND patients for sHLA-I (P < 0.02), greater in RRMS than in OIND and in NIND for sHLA-G (P < 0.001 and P < 0.01, respectively) and lower in RRMS than in OIND and in NIND for sFas (P < 0.001 and P < 0.02, respectively). An increase in CSF levels was identified in MRI active RRMS for sHLA-I (P < 0.01) and in MRI stable RRMS for sHLA-G (P < 0.01), whereas CSF values of sFas were decreased in RRMS without Gd-enhancing lesions (P < 0.02). In MS patients with no evidence of MRI disease activity, a trend towards an inverse correlation was found between CSF concentrations of sHLA-G and sHLA-I and between CSF levels of sHLA-G and sFas. Our results indicate that enhanced CSF levels of sHLA-I antigens most likely represent an indirect manifestation of intrathecal immune activation taking place in neuroinflammation. Conversely, reciprocal fluctuations in CSF sHLA-G and sFas levels observed when MRI disease activity resolved suggest that sHLA-G could play an immunomodulatory role in MS through Fas/FasL-mediated mechanisms.

Cerebral blood flow (CBF) alterations following post-traumatic contusions have been demonstrated in recent papers. We evaluated regional CBF (rCBF) by means of Xenon-enhanced computerized tomography (Xe-CT) in 29 traumatic intracerebral hematomas, from 22 patients with severe head injury (GCS < or = 8). Fifty traumatic hematoma/Xe-CT CBF measurements were obtained from 39 Xe-CT studies performed during the acute phase (corresponding to the first 20 days post-injury). The rCBF was measured in three different regions of interest: the hemorrhagic core, the perihematoma edematous low-density area, and a 1-cm rim of perihematoma normal-appearing brain tissue, surrounding the edematous low-density area. We found a centrifugal improvement of rCBF as well as a decrease in the rates of CBF levels below 18 mL/100 g/min from the core to the periphery (p < 0.0001), which persisted over time. Ischemic rCBF values were detected in the perihematoma low-density area only in 24% of the traumatic hematomas. The time course of rCBF levels showed a reduced flow in the first 24 h, with a recovery of flow from day 2 to day 4, followed by another reduced flow (p < or = 0.0001) both in the perihematoma edematous low-density area and in the non-lesioned tissue. Our findings suggest that the only area with persistent ischemic values was the hemorrhagic core. Low rCBF levels seen in the perihematoma low-density area may only be ascribed partially to ischemia and can possibly recover over time. These results could encourage a surgical approach based on an early evacuation of the hemorrhagic core associated to a preservation of the surrounding edematous tissue.

We have investigated the presence of non-classical soluble HLA-G molecules (sHLA-G) in cerebrospinal fluid (CSF) of multiple sclerosis (MS) patients and the possible relationships between CSF levels of sHLA-G, classical soluble HLA-I (sHLA-I) molecules, IL-10 amounts and Magnetic Resonance Imaging (MRI) findings were evaluated. We studied by ELISA technique the sHLA-I, sHLA-G and IL-10 levels in CSF of 50 relapsing-remitting (RR) MS patients stratified according to clinical and MRI evidence of disease activity. Thirty-six patients with other inflammatory neurological disorders (OIND) and 41 with non-inflammatory neurological disorders (NIND) were used as controls. CSF mean levels were significantly higher in MS and OIND than in NIND for sHLA-I (p<0.001) and in MS than in controls for sHLA-G (p<0.001), with no differences among the various groups for IL-10 mean concentrations. An increase in CSF sHLA-I was found in MS patients with Gd-enhancing lesions (p<0.01), while sHLA-G and IL-10 were more represented in MS patients without lesional activity on MRI scans (p<0.02). In MRI-inactive MS, CSF IL-10 mean concentrations were significantly greater in patients with CSF-detectable levels of sHLA-G than in those without any evidence of CSF sHLA-G expression (p<0.05). Our findings suggest that CSF classical sHLA-I and non-classical sHLA-G levels may modulate MS activity as assessed by MRI acting in opposite directions. The association observed between sHLA-G and IL-10 when Gd-enhancing lesion resolved indicates a potential immunoregulatory role for IL-10 in the control of MS disease activity by shifting the sHLA-I/sHLA-G balance towards sHLA-G response.